Aloe-emodin from rhubarb (Rheum rhabarbarum) inhibits lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages

Ethnopharmacological relevance

Rheum rhabarbarum (rhubarb) has long been used for the treatment of inflammation in China and other Asian countries. However, the mechanism underlying the anti-inflammatory activity of this medicinal plant is not fully understood. The present study was designed to investigate the anti-inflammatory effects of anthraquinones, the major constituents in rhubarb, and the molecular mechanism involved in their anti-inflammatory effects.

Materials and methods

RAW264.7 cells were stimulated by lipopolysaccharide (LPS) in the presence or absence of the compounds examined. The proliferation of RAW264.7 cells was assayed by the Alamar-Blue method. The quantity of nitric oxide (NO) was determined by Griess assay. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR. Inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor κBα (IκBα), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and Akt/phosphoinositide 3-kinase (PI3K) protein expression levels were determined by Western blotting.

Results

Aloe-emodin markedly suppressed the production of NO, interleukin-6 (IL-6), and interleukin-1β (IL-1β) in LPS-stimulated RAW264.7 cells with no apparent cytotoxicity. The mRNA expression levels of iNOS, IL-6, and IL-1β genes were also significantly inhibited by aloe-emodin. Western blot analysis showed that aloe-emodin suppressed LPS-induced iNOS protein expression, IκBα degradation, and the phosphorylation of ERK, p38, JNK, and Akt.

Conclusions

These results demonstrate that aloe-emodin is the bioactive component of rhubarb that confers an anti-inflammatory effect through a likely mechanism involving a decrease in pro-inflammatory cytokine production in LPS-induced RAW264.7 macrophages via inhibition of NF-κB, MAPK, and PI3K pathways.     

Effects of taraxasterol on inflammatory responses in lipopolysaccharide-induced RAW 264.7 macrophages

Aim of the study

Taraxasterol, a pentacyclic-triterpene, was isolated from the Chinese medicinal herb Taraxacum officinale. In the present study, we investigated the in vitro anti-inflammatory activity of taraxasterol in lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages.

Materials and methods

RAW 264.7 cells were pretreated with 2.5, 5, or 12.5 μg/ml of taraxasterol 1 h prior to treatment with 1 μg/ml of LPS. Nitric oxide (NO) level in supernatants from cells was examined by Griess reaction, the concentrations of prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured by ELISA. Nuclear factor kappa B (NF-κB) activation was evaluated by immunocytochemical analysis.

Results

We found that taraxasterol inhibited NO, PGE₂, TNF-α, IL-1β and IL-6 production in LPS-induced RAW 264.7 macrophages in a dose-dependent manner. Further studies revealed that taraxasterol prevented the LPS-induced NF-κB translocation from cytoplasm into nuclear.

Conclusions

These results indicate that taraxasterol has anti-inflammatory effect by blocking NF-κB pathway.     

Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW 264.7) and in experimental animals

Ethnopharmacological relevance

The rhizomes of Kaempferia parviflora Wall. ex Baker have been used in Thailand for treatment of gout, apthous ulcer, peptic ulcer and abscesses.

Aim of the study

In our previous study, the crude ethanol extract of Kaempferia parviflora and its compound (5, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone), was reported to show nitric oxide (NO) inhibition in RAW 264.7 cells. The present study is thus investigated the anti-inflammatory mechanism of Kaempferia parviflora extract and compound 5 against inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expressions.

Materials and methods

The extract of Kaempferia parviflora and its compound were tested against NO and prostaglandin E₂ (PGE₂) releases using RAW264.7 cells as well as studied on anti-inflammatory activity in carrageenan-induced rat paw edema and acute toxicity in mice.

Results

The results revealed that the ethanol extract of Kaempferia parviflora markedly inhibited PGE₂ release with an IC₅₀ value of 9.2 μg/ml. This plant extract and compound 5 also suppressed mRNA expression of iNOS in dose-dependent manners, whereas COX-2 mRNA expression was partly affected. According to the in vivo study, chloroform and hexane fractions greater decreased rat paw edema than ethanol, ethyl acetate and water fractions.

Conclusion

The mechanisms for anti-inflammatory activity of Kaempferia parviflora and compound 5 are mainly due to the inhibition of iNOS mRNA expression but partly through that of COX-2 mRNA.     

Suppression of lipopolysaccharide-induced of inducible nitric oxide synthase and cyclooxygenase-2 by Sanguis Draconis, a dragon's blood resin, in RAW 264.7 cells

Sanguis Draconis (SD) is a kind of dragon's blood resin that is obtained from Daemomorops draco (Palmae). It is used in traditional medicine and has shown anti-inflammatory activity in some diseases. In this study, we examined the effects of Sanguis Dranonis ethanol extract (SDEE) on LPS-induced inflammation using RAW 264.7 cells. Our data indicated that SDEE inhibits LPS-stimulated NO, PGE2, IL-1 beta and TNF-alpha release, and iNOS and COX-2 expression. Furthermore, SDEE suppressed the LPS-induced p65 expression of NF-kappa B, which was associated with the inhibition of I kappa B-alpha degradation. We also found that the expression of HO-1 was significantly increased in RAW 264.7 cells by SDEE. These results suggest among possibilities of anti-inflammation that SDEE inhibits the production of NO and PGE2 by the down-regulation of iNOS and COX-2 gene expression via the suppression of NF-kappaB (p65) activation. SDEE can induce HO-1 over-expression in macrophage cells, which indicates that it may possess antioxidant properties. This result means that SEDD its anti-inflammatory effects in macrophages may be through a novel mechanism that involves the action of HO-1. Thus, SD could provide a potential therapeutic approach for inflammation-associated disorders.     

A fraction of stem bark extract of Entada africana suppresses lipopolysaccharide-induced inflammation in RAW 264.7 cells

Ethnopharmacological relevance

Entada africana is a plant used in African traditional medicine for the treatment of stomachache, fever, liver related diseases, wound healing, cataract and dysentery.

Aims of the study

This study aimed at evaluating the anti-inflammatory activity of fractions of the stem bark extract of the plant using lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages model.

Materials and methods

The crude extract was prepared using the mixture CH₂Cl₂/MeOH (1:1, v/v) and fractionated by flash chromatography using solvents of increasing polarity to obtain five different fractions. The effects of the fractions on the cells viability were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and their inhibitory activity against LPS-induced nitric oxide (NO) production screened by Griess test. The most active fraction was further investigated for its effects on reactive oxygen species (ROS) production using flux cytometry, the expression of inducible nitric oxide synthase (iNOS), pro-and anti-inflammatory cytokines (IL1β, TNFα, IL6, IL10 and IL13) by RT-PCR, and the activity of the enzyme p38 MAPK kinase by enzyme-linked immunosorbent assay (ELISA).

Results

The fractions presented no significant effect on the viability of macrophages at 100 μg/ml after 24 h incubation. The CH₂Cl₂/MeOH 5% (Ea5) fraction was found to be the most potent in inhibiting NO production with a half inhibition concentration (IC₅₀)=18.36 μg/ml, and showed the highest inhibition percentage (89.068%) in comparison with Baicalin (63.34%), an external standard at 50 μg/ml. Ea5, as well as Baicalin significantly (P<0.05) inhibited the expression of TNFα, IL6 and IL1β mRNA, attenuated mRNA expression of inducible NO synthase in a concentration-dependent manner, stimulated the expression of anti-inflammatory cytokines (IL10 and IL13), and showed a 30% inhibition of the activity of p38 MAPK kinase.

Conclusion

The results of the present study indicate that the fraction Ea5 of Entada africana possesses most potent in vitro anti-inflammatory activity and may contain compounds useful as a therapeutic agent in the treatment of inflammatory related diseases cause by over-activation of macrophages.     

Gleditsia sinensis thorns inhibit the production of NO through NF-kappaB suppression in LPS-stimulated macrophages

The thorns of Gleditsia sinensis LAM. (Leguminosae) have been used in traditional medicine for the treatment of inflammatory diseases including swelling, suppuration, carbuncle and skin diseases in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of Gleditsia sinensis thorns in RAW 264.7 macrophages. The aqueous extract of Gleditsia sinensis thorns (AEGS) inhibited LPS-induced NO secretion as well as inducible nitric oxide synthase (iNOS) expression, without affecting cell viability. Furthermore, AEGS suppressed LPS-induced NF-kappaB activation, phosphorylation and degradation of IkappaB-alpha, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). These results suggest that AEGS has the inhibitory effects on LPS-induced NO production and iNOS expression in macrophages through blockade in the phosphorylation of MAPKs, following IkappaB-alpha degradation and NF-kappaB activation.     

Suppression of lipopolysaccharide-induced inflammatory responses in RAW 264.7 murine macrophages by aqueous extract of Clinopodium vulgare L. (Lamiaceae)

Ethnopharmacological relevance

The wild basil Clinopodium vulgare L. is commonly used in Bulgarian folk medicine for treatment of irritated skin, mastitis- and prostatitis-related swelling, as well as for some disorders accompanied with significant degree of inflammation (e.g. gastric ulcers, diabetes, and cancer).

Aim of study

To determine the effect of aqueous extract of Clinopodium vulgare L. on LPS-induced inflammatory responses of murine RAW 264.7 macrophages.

Materials and methods

Cell cytotoxicity was evaluated by MTT assay. Protein expression levels were monitored by Western blot analysis. Production of NO and PGE₂ was measured by the Griess colorimetric method and enzyme immunoassay, respectively. Activation of MMP-9 was visualized by gelatin zymography. Cytokine levels were determined by BioPlex assay. Intracellular ROS and free radical scavenging potential were measured by DCFH-DA and DPPH method, respectively. Xanthine oxidase activity was evaluated spectrophotometrically.

Results

The extract suppresses NF-κB activation by preventing Iκ-B phosphorylation and inhibits the phosphorylation of p38 and SAPK/JNK MAPKs. It down-regulates iNOS expression which manifests as a drastic decrease of NO production, inhibits MMP-9 activation, but does not affect COX-2 protein levels and reduces only slightly the released PGE₂. Secretion of IL-1β and Il-10 is greatly reduced, whereas suppression of TNF-α and GM-CSF production is less dramatic. The extract has strong free radical scavenging properties and exerts inhibitory effect on xanthine oxidase activity, which lowers the levels of intracellular ROS.

Conclusion

The study provides evidence for the anti-inflammatory potential of Clinopodium vulgare L. aqueous extract.     

Canna indica L. attenuates high-glucose- and lipopolysaccharide-induced inflammatory mediators in monocyte/macrophage

Ethnopharmacological relevance

Canna indica L. (CI) has been widely used as a folklore medicine in tropical and subtropical areas with beneficial effects in numerous diseases, including infection, rheumatism, hepatitis, and it has also been identified as an antioxidant.

Materials and methods

The present study aimed to investigate the effect of Canna indica CI ethanolic extract (CIE) on productions of nitric oxide (NO), prostaglandin E₂ (PGE₂), and interleukin-1β (IL-1β) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. In addition, the effects of CIE in high glucose (HG)-induced U937 monocytes on mRNA expressions of IL-8 and monocyte chemoattractant protein-1 (MCP-1), and regulation of mitogen-activated protein kinase (MAPK) pathways were also identified.

Results

CIE was found to inhibit the production of inflammatory mediators including NO, IL-1β, and PGE₂ from LPS-induced RAW 264.7 macrophages. The increases in HG-induced mRNA expressions of IL-8 and MCP-1 were also significantly inhibited by CIE. Stimulation of HG in U937 monocytes resulted in activation of p38 MAPK, ERK1/2, and JNK. However, CIE treatment significantly decreased phosphorylation of p38 MAPK, ERK1/2, and JNK.

Conclusion

The present study demonstrated that CIE suppressed the LPS-induced inflammatory mediator production and also inhibited HG-induced inflammatory mediator expression by the regulation of MAPK pathway.     

Suppressive effects of wild bitter gourd (Momordica charantia Linn. var. abbreviata ser.) fruit extracts on inflammatory responses in RAW 264.7 macrophages

Aim of the study

Bitter gourd (Momordica charantia) is used to treat various diseases including inflammation. A wild species of bitter gourd, Momordica charantia Linn. var. abbreviata ser. (WBG), is considered to be more potent in disease prevention than is bitter gourd; however, little is known about the biological and physiological characteristics of WBG.

Materials and methods

The present study investigated the anti-inflammatory effect of WBG on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Results

Among the hot water, 95% ethanol, and ethyl acetate extracts of WBG, the ethanol extract showed the greatest reduction of LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production and inducible nitric oxide synthase (iNOS) and pro-interleukin-1β expression. LPS-induced cyclooxygenase-2 expression was not affected by WBG extracts. Compared with WBG, extracts from bitter gourd showed a lesser inhibition of LPS-induced events. Electrophoretic mobility shift assay further showed that both the hot water and the ethanol extracts of WBG inhibited NF-κB activation. Although information is lacking on the bioactive components of WBG, the phenolic compound contents of each extract significantly paralleled its anti-inflammatory ability (r = 0.74, 0.88 and 0.65 for NO, PGE₂ and iNOS expression, respectively, P < 0.05).

Conclusions

These results suggest that WBG is beneficial for reducing LPS-induced inflammatory responses by modulating NF-κB activation.     

Myrsine seguinii ethanolic extract and its active component quercetin inhibit macrophage activation and peritonitis induced by LPS by targeting to Syk/Src/IRAK-1

Ethnopharmacological relevance

Myrsine seguinii H. LÉVEILLÉ (syn. Rapanea neriifolia) (Myrsinaceae) is a medicinal plants traditionally used in Myanmar to treat infectious and inflammatory diseases. Since none of reports have systematically demonstrated the anti-inflammatory activity of this plant, we aimed to mechanistically understand the regulatory roles of the plant in inflammatory responses using the ethanolic extract of Myrsine seguinii (Ms-EE).

Materials and methods

Activated macrophages and peritonitis symptoms induced by lipopolysaccharide (LPS) were employed. HPLC analysis was used to identify active components. To characterize direct target enzymes, kinase assay was established.

Results

Ms-EE inhibited the production of nitric oxide (NO) and prostaglandin (PG)E₂ in RAW264.7 cells and peritoneal macrophages stimulated by LPS. This extract suppressed the mRNA expression of the inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 genes by down-regulating the activation of nuclear factor (NF)-κB and activator protein (AP-1). Interestingly, it was found that Ms-EE can directly suppress the enzyme activities of Syk, Src, and interleukin-1 receptor-associated kinase-1 (IRAK-1). Similarly, orally administered Ms-EE inhibited the phosphorylation of Src and Syk in peritoneal exudate-derived cells prepared from peritonitis. Finally, HPLC analysis clearly demonstrated that quercetin is a major active component with suppressing activity on the release of inflammatory mediators (NO and PGE₂), and the enzyme activities of Src, Syk, and IRAK-1.

Conclusion

Ms-EE containing quercetin negatively modulates macrophage-mediated in vitro inflammatory responses and LPS-induced peritonitis by blocking the Src/Syk/NF-κB and IRAK-1/AP-1 pathways, which contributes to its major ethnopharmacological use as an anti-inflammatory herbal medicine.     

HMCO5, herbal extract, inhibits NF-kappaB expression in lipopolysaccharide treated macrophages and reduces atherosclerotic lesions in cholesterol fed mice

HMCO5 is a herbal extract which comprises of eight different herbs. We studied whether this extract has anti-atherosclerotic effects. In lipopolysaccharide (LPS) stimulated RAW264.7 cells, HMCO5 inhibited NF-kappaB activation as well as iNOS promoter activity, inhibited the secretion of TNF-alpha and IL-1beta, and directly inhibited the intracellular accumulation of reactive oxygen species. ApoE knock-out mice fed a high-fat high-cholesterol diet with HMCO5 for 10 weeks showed a significant reduction in atherosclerotic lesions. A notable finding was the preservation of the smooth muscle cell layer in the media of aorta in the HMCO5 co-treated mice. HMCO5 treated mice did not show significant decrease in serum level of cholesterol. These results suggest that HMCO5 has anti-atherosclerotic effects which in part may be attributable to the inhibition of production of NF-kappaB dependent pro-inflammatory cytokines.     

Inula japonica extract inhibits mast cell-mediated allergic reaction and mast cell activation

Ethnopharmacological relevance

The flowers of Inula japonica (Inulae Flos) have long been used in traditional medicine for the treatment of bronchitis, digestive disorders, and inflammation. However, the mechanisms underlying its anti-inflammatory effects remain yet to be elucidated. The objectives of this study were 1) to assess the anti-allergic activity of the ethanol extract of flowers of Inula japonica extract (IFE) in vivo, 2) to investigate the mechanism of its action on mast cells in vitro, and 3) to identify its major phytochemical compositions.

Materials and methods

The anti-allergic activity of IFE was evaluated using mouse bone marrow-derived mast cells (BMMCs) in vitro and a passive cutaneous anaphylaxis (PCA) animal model in vivo. The effects of IFE on mast cell activation were evaluated in terms of degranulation, eicosanoid generation, Ca²⁺ influx, and immunoblotting of various signaling molecules.

Results

IFE inhibited degranulation and the generation of eicosanoids (PGD₂ and LTC₄) in stem cell factor (SCF)-stimulated BMMCs. Biochemical analysis of the SCF-mediated signaling pathways demonstrated that IFE inhibited the activation of multiple downstream signaling processes including mobilization of intracellular Ca²⁺ and phosphorylation of the mitogen-activated protein kinases (MAPKs), PLCγ1, and cPLA₂ pathways. When administered orally, IFE attenuated the mast cell-mediated PCA reaction in IgE-sensitized mice. Its major phytochemical composition included three sesquiterpenes, 1-O-acetylbritannilactone, britanin and tomentosin.

Conclusions

This study suggests that IFE modulates eicosanoids generation and degranulation through the suppression of SCF-mediated signaling pathways that would be beneficial for the prevention of allergic inflammatory diseases. Anti-allergic activity of IFE may be in part attributed particularly to the presence of britanin and tomentosin as major components evidenced by a HPLC analysis.     

Atropa acuminata Royle Ex Lindl. blunts production of pro-inflammatory mediators eicosanoids., leukotrienes,cytokines in vitro and in vivo models of acute inflammatory responses

Ethnopharmacological relevance

Atropa acuminata Royle Ex Lindl. has been widely used in folk medicine for several inflammatory disorders such as arthritis, asthma, conjunctivitis, encephalitis, pancreatitis, peritonitis, acute infections and neuroinflammatory disorders.

Aim of the study

Our aim was to evaluate Atropa acuminata for its anti-inflammatory properties and to delineate its possible mechanism of action on the modulation of the inflammatory mediators.

Materials and methods

We investigated the inhibitory action of ethanolic extract of Atropa acuminata (AAEE) on production of NO, TNF-α and IL-1β in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and also assayed it for COX 1/2 and 5-LOX inhibitory activities. Next AAEE was tested in acute inflammatory animal models., carragenean induced rat paw edema, carragenean induce pleurisy in rats and vascular permeability in mice and the effects on NO, PGE₂ and LTB₄ production in the pleural fluid and paw exudates were evaluated. In addition the effects on leukocyte migration and exudation and vascular permeability were also observed.

Results

Our findings summarized novel anti-inflammatory mechanisms for Atropa acuminata based on dual in vitro cyclooxygenase 1/2/ and 5-Lipoxygenase inhibitory activities and also significant downregulation of nitric oxide (NO) and pro-inflammatory cytokin (TNF-α and Il-1 β) release in LPS-stimulated RAW 246.7 macrophage cell line. In acute inflammatory models in vivo (carragenean induced edema, carragenean induced pleurisy in rats and vascular permeability in mice), AAEE exhibited an extensive diverse mechanism for anti-inflammatory properties. This was indicated on the basis of dose dependent suppression of multi targeted inflammatory mediators., NO, TNF-α and IL-1β, eicosanoids., PGE₂ and leukotrienes., LTB₄ along with significantly decreased leucocyte migration, exudation and decreased vascular permeability. These effects were more potent and prolonged than traditional NSAIDS, thereby indicating fewer side effects. AAEE was found to be safe for long term administration, as confirmed by the results of acute toxicity studies and MTT assay. The complex mode of action of the herbs was attributed possibly due to the high polyphenolic, flavanol and flavonoid content present in the extracts as observed by means of quantitative screening for phytochemicals.

Conclusion

Our study provides scientific evidence to support the traditional anti-inflammatory uses of Atropa acuminata and is probably due to inhibitory effects on multiple inflammatory mediators which indicates a promising potential for the development of a strong anti-inflammatory agent from this plant.